The development of vestibulocochlear efferents and cochlear afferents in mice

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Pergamon

Int. J. Devl Neuroscience, Vol. 15, No. 4/5, pp. 671-692, 1997

Copyright © 1997 ISDN. Published by ElsevierScienceLtd Printed in Great Britain. All rights reserved 0736-5748/97 $17.00+0.00

Plh S0736-5748(96)00120-7 THE

DEVELOPMENT OF VESTIBULOCOCHLEAR EFFERENTS AND COCHLEAR AFFERENTS IN MICE L. L. BRUCE, J. KINGSLEY, D. H. NICHOLS and B. FRITZSCH* Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, U.S.A.

Abstract--Wehave reinvestigated the embryonic development of the vestibulocochlear system in mice using anterograde and retrograde tracing techniques. Our studies reveal that rhombomeres 4 and 5 include five motor neuron populations. One of these, the abducens nucleus, will not be dealt with here. Rhombomere 4 gives rise to three of the remaining populations: the facial branchial motor neurons; the vestibular efferents; and the cochlear efferents. The migration of the facial branchial motor neurons away from the otic efferents is completed by 13.5 days post coitum (dpc). Subsequently the otic efferents separate into the vestibular and cochlear efferents, and complete their migration by 14.5 dpc. In addition to their common origin, all three populations have perikarya that migrate via translocation through secondary processes, form a continuous column upon completion of their migrations, and form axonal tracts that run in the internal facial genu. Some otic efferent axons travel with the facial branchial motor nerve from the internal facial genu and exit the brain with that nerve. These data suggest that facial branchial motor neurons and otic efferents are derived from a common precursor population and use similar cues for pathway recognition within the brain. In contrast, rhombomere 5 gives rise to the fourth population to be considered here, the superior salivatory nucleus, a visceral motor neuron group. Other differences between this group and those derived from rhombomere 4 include perikaryal migration as a result of translocation first through primary processes and only then through secondary processes, a final location lateral to the branchial motor/otic efferent column, and axonal tracts that are completely segregated from those of the facial branchial and otic efferents throughout their course inside the brain. Analysis of the peripheral distribution of the cochlear efferents and afferents show that efferents reach the spiral ganglion at 12.5 dpc when postmitotic ganglion cells are migrating away from the cochlear anlage. The efferents begin to form the intraganglionic spiral bundle by 14.5 dpc and the inner spiral bundle by 16.5 dpc in the basal turn. They have extensive collaterals among supporting cells of the greater epithelial ridge from 16.5 dpc onwards. Afferents and efferents in the basal turn of the cochlea extend through all three rows of outer hair cells by 18.5 dpc. Selective labeling of afferent fibers at 20.5 dpc (postnatal day 1) shows that although some afferents are still in early developmental stages, some type II spiral ganglion cells already extend for long distances along the outer hair cells, and some type I spiral ganglion cells end on a single inner hair cell. These data support previous evidence that in mice the early outgrowth of afferent and efferent fibers is essentially achieved by birth. 4) 1997 ISDN Key words: cochlear development; synaptogenesis; afferents; efferents.

The development of the vestibulocochlear system has been extensively investigated in a variety of mammals. Early investigations based on uttrastructural morphology42 provided the conceptual framework for cochlear synaptogenesis upon which most studies of cochlear development have been based for over 20 years. Recently, the application of modern labeling techniques to rodents, and mice in particular, has challenged some of the hypotheses within this framework. Investigators have often hypothesized that efferent fibers reach the cochlea several days after afferents.42 Once efferents arrive at the inner hair cells, they extend to the outer hair cells only after the withdrawal of a temporary extension of type I afferents to the outer hair cells. 42 Although these hypotheses appeared to fit most of the data available at that time, more recent evidence has shown that the arrival of efferent fibers at the inner and outer hair cells is much earlier then previously shown. Based on histochemical, immunocytochernical and ultrastructural data, Sobkowicz and collaborators 52 found that efferents first arrive at inner hair cells at the base of the cochlea as early as 4 days prior to birth. Subsequently the efferents extend to outer hair cells in late embryos and early neonates in a basal to apical progression. Recent analyses using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and dextran amine labeling in mouse embryos show that the first efferent axons reach the spiral ganglion at 12 days post coitum (dpc). 17Thus efferent fibers appear to reach the differentiating spiral

* To whom all correspondence should be addressed. 671

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ganglia and at least the inner hair cells several days prior to birth in mice. It is unknown what these fibers do during the several days before they finally synapse on hair cells. However, data in postnatal rats suggest that efferents arrive at inner and outer hair cells several days prior to the appearance of ultrastructurally identifiable synapses.5'9 The origin and differentiation of the olivocochlear neurons has been identified in parallel studies. The olivocochlear efferent neurons and the facial branchial motor neurons of mice become postmitotic between 10.5 and 12 dpc (11.5 14 dpc in rats) ~, and temporarily remain in the same area of the hindbrain (rhombomere 4) from which they are both derived.~7 Subsequently these cells disperse through differential migration into different areas of the hindbrain. The segregation of the facial branchial motor neurons from the otic (vestibulocochlear) efferents has been described in whole mounts and appears to be complete by 13.5 dpc.16 To date, several details of the migration patterns that produce this segregation have not been elucidated, including the segregation of otic efferents into vestibular and cochlear efferent populations and the ontogenetic relationships between the facial visceral motor neurons of the superior salivatory nucleus and otic efferents. The aim of this paper is twofold. First, we will: 1. describe the segregation of vestibulocochlear efferent neurons from facial branchial motor neurons; 2. provide evidence that vestibulocochlear efferents and preganglionic parasympathetic (visceral) motor neurons arise from separate populations; and 3. demonstrate when and how vestibulocochlear efferents segregate into distinct vestibular and cochlear efferent groups. Second, we will: 1. describe how efferent fibers grow towards the developing cochlea; and 2. compare the progression of afferents and efferents toward and within the cochlea by selectively filling only afferents to one cochlea and only efferents to the other cochlea in the same animal.

EXPERIMENTAL PROCEDURES Animals

We have used two groups of mouse embryos. The first 220 ranged in age from 12 dpc to newborn (ca 20 dpc). We define noon, 12:00, following the appearance of the vaginal plug as 0.5 dpc, and day

of birth as postnatal day 0 (P0). Later ages were calculated from that time. These embryos were collected after cervical dislocation of the mother and were either immediately immersed or perfused (young embryos) or anesthetized with a lethal dose of Beuthanasia-D [900 mg/kg of sodium pentobarbital, intraperitoneally (i.p.); embryos of 18.5 dpc and older] and then perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) with 0.5 mM EDTA injected into the ventricle. A second group of 32 embryos between ages 11.5-12.5 dpc were used for in vitro staining of the nerve fibers and/or neuronal cell bodies with dextran amines. ~5 We used three different mouse strains, CF1, BALB/c, and C3H mice to minimize the possibility of error due to any unrecognized auditory condition not yet fully described in one of these strains. All procedures are approved by the IACUC committee of Creighton University. D i I labeling

The heads of fixed embryos or neonates were either divided into two along the sagittal plane or one ear was exposed. Nylon filter strips soaked in a solution of DiI and dimethylformamide~7were inserted into the cochlear nucleus complex (dorsal and ventral cochlear nuclei) of one half brain, and into the efferent bundle near the floor plate of the other half brain. In whole embryo heads, DiI was inserted into one ear (e.g. the left cochlea) or into the facial nerve (either the greater petrosal nerve or the facial nerve distal to the geniculate ganglion). A schematic drawing demonstrating the various application sites used to label motor neurons is shown in Fig. 1. Heads were incubated for 2-6days at 37°C in an oven (depending on age and thus distance required for diffusion) and subsequently analyzed.

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(; OFacial BMN • t i" II Sup. Saliv.N. Otic Efferents. Fig. 1. Schematic drawing indicating the different application sites used to label various groups of efferent nerves. Dye applications were placed in: (1) the greater petrosal nerve (GP), which would label a subpopulation of the superior salivatory nucleus; (2) the facial nerve distal to the greater petrosal nerve and proximal to the divergence of the chorda tympani nerve (CT), which would label all of the facial branchial

motor neurons and a portion of the superior salivatory nucleus; (3) the inner ear or cranial nerve VIII, which would label the anterior (VIIIa) and posterior (VIIIp) rami and would label cochlear and vestibular efferents; (4) the inner ear, facial, and intermediate nerves, which would label all facial, vestibulocochlear, and superior salivatory neurons; and (5) the brainstem near the midline, which would label facial branchial motor neurons and vestibulocochlear efferents ipsilaterally and the vestibulocochlear efferents contralaterally. To analyze the peripheral fiber organization, the contralateral inner ear (in cases of ear applications) or the ipsilateral inner ear (in cases of central efferent bundle or afferent applications) was dissected and flat-mounted on a slide to reveal the distribution of all efferents and afferents (central applications) as compared to bilaterally projecting efferents (ear applications). In cases with a facial nerve application we also checked the distribution of fibers to the ipsilateral and contralateral inner ear and otic region in whole mounts and/or sections. Brains were analyzed either as whole mounts or as vibratomed sections (80-100 gm thick, transverse sections). Sections were obtained by first embedding the brains or whole heads in 10% warm gelatin which was then hardened in 10% paraformaldehyde for at least 12hr at 4°C. Vibratome sections were collected in 0.1 M phosphate buffer (pH 7.4), mounted in F l u o r o m o u n t (Fisher) and viewed, photographed, and analyzed in a compound microscope (Olympus BH2 or Nikon Optiphot) using epifluorescent illumination and a standard rhodamine or Texas red filter cube. In addition some images were processed using ImagePro software and a deconvolution algorithm to clean up the haze due to out-of-focus structures (electronic confocal imaging; Vaytek, Iowa City). For photographic documentation we used TechPan film (Kodak) developed with Liquidol to obtain fine grained images. Some preparations were photoconverted, embedded in epoxy resin, and either examined as whole mounts or sectioned for a more detailed analysis. 59 We rarely observed transneuronal diffusion of Di! in these experiments, because we focused on developmental events prior to synaptogenesis, and prior to the time that m e m b r a n e - m e m b r a n e contacts would be made between

neurons.4 Dextran labeling Embryos between 11.5 and 12.5 dpc were used for tracing of efferent fibers from the brainstem or for retrogradely labeling efferent perikarya from the periphery. Fluorescent (Texas red, fluorescein) and/or biotin labeled 3 k D a dextran amines (Molecular Probes) were used as previously described. ~5 Briefly, embryos were kept in cold (4°C) Neurobasal medium (Gibco). Heads were dissected so that either the fourth ventricle was exposed (for central applications) or the inner ear and/or facial nerve (for peripheral applications). The dextrans were applied for 10 sec to either a cut in the brainstem (rhombomere 4) or to the ear and/or the facial nerve. After the applications, the preparations were washed and incubated for 2 hr at 4°C. Subsequently the preparations were fixed in 4% paraformaldehyde (fluorescent dextran amines) or 2.5% glutaraldehyde (biotin dextran amines). Brains and inner ears were analyzed as whole mounts. These whole mounts were reacted with ABC

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complex and developed with diaminobenzidine (DAB) to visualize the biotin dextran amines. All whole mounts were coverslipped and examined with a compound microscope.

Orientation The hindbrain will herein be described as a flat mount opened along the dorsal midline and laid flat like a book. In this orientation, the floor plate is the midline reference structure and the direction thus passes from the floor plate through first the basal plate, then the sulcus limitans and finally the alar plate. The upper-most surface of the mount is the dorsal (ventricular) surface and the lowermost, the ventral (meningeal) surface. RESULTS This section will first describe the distribution of the retrogradely labeled cells and axonal collaterals following dye applications to different peripheral nerves. Second, the distribution of axons projecting to the ear following selective DiI applications to efferents (by applications within the brainstem, contralateral ear, and the ipsilateral facial nerve), or to afferents (by applications to the cochlear nuclei) will be described.

Retrograde labeling of cell bodies after peripheral applications As early as 10.5 dpc, we were able to label motor neurons after selective applications of either DiI or dextran amines to the facial nerve. From 11.5 dpc onward the otic efferent motor neurons were also labeled after selective applications to the greater petrosal nerve, facial nerve, or to the VIIIth nerve. Greater petrosal nerve labeling The motor component of the greater petrosal nerve arises from the superior salivatory nucleus which is the source of the preganglionic parasympathetic motor neurons that control secretion of the lacrimal, sublingual, and submaxillary glands. The greater petrosal nerve runs along the edge of the trigeminal ganglion towards the sphenopalatine ganglion. We applied label to this nerve together with the trigeminal nerve. From 10.5 dpc on, these applications consistently labeled fibers in the trigeminal nerve that could be followed to labeled trigeminal motor neurons in rhombomeres 2 and 3, and labeled fibers in the (facial) intermediate nerve that could be followed to the superior salivatory nucleus in rhombomere 5; neurons in rhombomere 4 were never labeled. By 10.5 dpc superior salivatory axons had grown laterally towards the sulcus limitans to exit the central nervous system in the intermediate nerve and from there joined the greater petrosal nerve, where label was applied. The labeled neurons within rhombomere 5 first appeared near the floor plate, and were apparently derived from that area. By 12 dpc (Fig. 2A and B) some superior salivatory perikarya had translocated laterally, through their axons, as far as the sulcus limitans. Once the perikarya reached the sulcus limitans, these cells formed a secondary process that extended ventrally towards the meninges. By 13.5dpc these cells had segregated into two groups of labeled cells, a smaller dorsal group near the ventricle and a larger ventral population near the meninges (Fig. 3A-D). The axons from both groups traveled rostrally to exit the central nervous system as part of the intermediate nerve. A similar population of neurons in rhombomere 5 was always labeled after applications to the entire facial nerve root (Figs 2A and B and 3C and D). Like those neurons projecting through the greater petrosal nerve, the population of neurons projecting through the facial nerve first formed axons that left the brain in the intermediate nerve, then their perikarya translocated laterally within their axons, and finally they extended a secondary process through which the perikarya migrated ventrally. By 13.5 dpc this latter population of neurons formed a cluster between the two clusters of cells that projected into the greater petrosal nerve, and were clearly distributed in the same dorso-ventral column as those projecting to the greater petrosal nerve (Fig. 3B and D). Facial nerve Two populations of cell bodies were labeled following DiI applications to the facial nerve (distal to the exit of the greater petrosal nerve) prior to 11 dpc: a small group of cells in rhombomere 5 and

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Fig. 2. The organization o f the three motor neuron groups o f rhombomeres 4 and 5 (r4, r5) and the early appearance of contralateral otic efferents and the internal facial genu/intermediate nerve organization. Figures depict flat mounts of brains at 11.5 dpc (A) and 12 dpc (B D), and a horizontal brain section at 15.5 dpc (E). Applications o f biotin dextran amine (A) or DiI (B-E) were made into the facial-otic nerve complex. Note that at 11.5 dpc most labeled neurons have not begun to migrate (A), but within I day many facial branchial motor neurons (fb) have translocated along the floor plate (fp) through r5, and have started to migrate laterally into r6 (B). Note also that during this period the visceral motor neurons of the future superior salivatory nucleus (ss) have moved from near the floor plate (A) to a more lateral position (B). The first contralateral otic efferent cell bodies (arrows) appear at 12 dpc, but labeled cells are never observed within the floor plate (C and D). By 15.5dpc (E) the internal facial genu (g) is fully formed and wraps around the abducens nucleus (a) [see also (A)] and around the laterally positioned ss which sends its axons into the intermediate nerve (in). Note the small fascicle of otic efferent fibers (ox) in r4 crossing the midline. Lines indicate the approximate positions of rhombomere boundaries. Bars = 100 ~tm.

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Fig. 3. Comparisons of nuclei projecting through the greater petrosal nerve and to the cochlea to those projecting through the facial nerve, including the chorda tympani nerve, and to the inner ear. Transverse 80 Ixm sections through the hindbrain (rhombomere 5) of 13.5 dpc mice. Dorsal is to the top in all figures. Medial is to the right in (A) and (B), and to the left in (C) and (D) after DiI applications to both the greater petrosal nerve and cochlea (A and B) and to the inner ear and facial nerve, including the chorda tympani nerve (C and D). Different parts of the superior salivatory nucleus (ss) are labeled after the two applications, depending on which peripheral visceral branch was labeled: visceral efferent neurons projecting to the greater petrosal nerve are more ventral (closer to the meninges) than those projecting to the chorda tympani. Note separate cell columns of the medially located facial branchial motor nucleus (fb) (C and D) and the laterally located visceral motor axons (A and B), and that the latter group turns to the intermediate nerve root (in) without contributing to the internal facial genu. Also note that these injections labeled afferent fibers from the cochlea [ca; (A)] or from the inner ear [ie; (C)], some of which extend towards the cerebellum [cb; (C)]. Bars = 100 ~tm.

a l a r g e r g r o u p o f cells in r h o m b o m e r e 4 (Fig. 2A). I n j e c t i o n s at o l d e r ages d e m o n s t r a t e d t h a t the small group becomes the superior salivatory component of the chorda tympani and the larger group b e c o m e s t h e facial b r a n c h i a l m o t o r n u c l e u s (Fig. 3 C a n d D). A s e a r l y as 11.0 d p c t h e first cells o f t h e l a r g e r g r o u p s t a r t to t r a n s l o c a t e t h e i r p e r i k a r y a , m i g r a t i n g c a u d a l l y , p a r a l l e l to t h e f l o o r plate, p a s t the a b d u c e n s m o t o r n e u r o n s o f r h o m b o m e r e 5, finally s t o p p i n g in t h e c a u d a l h a l f o f r h o m -

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bomere 5 and the rostral half of rhombomere 6 by 12.5 dpc (Fig. 2A and B). Once this migration is complete, these cells extend in a broad sweep from the floor plate ventrally towards the meninges. By 13.5 dpc they form a column situated medial and (mostly) caudal to the visceral motor neurons of the superior salivatory nucleus of rhombomere 5 (Fig. 3C and D). Thus, in transverse sections through the caudal half of rhombomere 5, the visceral superior salivatory motor neurons are lateral to the rostral-most branchial motor neurons (Figs 2E and 3C and D).

Otic efferents Selective dye applications to the otic (vestibulocochlear) vesicle or nerve between ages l 1.0 and 12.0dpc labeled a group of cells ipsilaterally in the same location as the facial branchial motor neurons in rhombomere 4. At 12.0 dpc we found that applications to the otic vesicle, even in animals from the same litter, would sometimes label no contralateral cells, a few contralateral cells, or many contralateral cells (Fig. 2C and D). We occasionally found labeled fibers tipped with growth cones in the floor plate of rhombomere 4. This variation between animals may reflect differences in developmental stages between fetuses within the same litter. However, by 12.5 dpc the presence of the contralateral cells is consistent after both central and peripheral injections (Fig. 4A and B). At 12.0 dpc dextran amines applied to a cut lateral to the floor plate that severed facial and otic efferent axons labeled a large mixed population of otic efferent and facial branchial motor neurons ipsilaterally and only otic efferents contralaterally (Fig. 4C). This sudden appearance of these contralateral cells suggests that axons (or axon collaterals) from these contralateral cells have grown across the floor plate of rhombomere 4, but have not consistently reached the otic vesicle at 12.0 dpc. The spindle-shaped motor neurons of both populations have two distinct orientations that can be identified as early as 11 dpc. The facial branchial motor neurons consistently orient their processes rostrocaudally, and the otic efferents orient theirs mediolaterally (Figs 2A and 4A). After 12.5 dpc there is a migration of the otic efferent neurons from the midline of rhombomere 4 laterally and caudally into rhombomeres 4 and 5 (Fig. 4A). Like the facial branchial motor neurons this migration is the result of a translocation through a secondary process, except that these cells migrate in a predominantly lateral direction away from the floor plate rather than caudally and parallel to it as do the facial branchial motor neurons. By 12.5 dpc, these migrations begin to form three distinct populations of cells on either side of the hindbrain (Fig. 4A): 1. A ventrolateral, longitudinal column of cells that is anterior and lateral to the facial branchial motor nucleus and projects predominantly to the ipsilateral periphery. 2. A dorsolateral group of cells that migrates laterally just beneath the dorsal (ventricular) surface and projects bilaterally to the periphery. 3. A densely clustered, dorsomedial group of cells that remains near the floor plate and projects bilaterally to the periphery. The segregation of these three groups is completed by 14.5 dpc. Applications to specific vestibular and cochlear sensory epithelia demonstrate the specific targets of these different cell groups. Applications in the vestibular epithelia label vestibular efferent cells in the dorsolateral and dorsomedial cell clusters (Fig. 5A-F). The dorsolateral cluster lies adjacent to the fourth ventricle and immediately dorsal to the point where the facial branchial motor fibers diverge from the otic efferents towards cranial nerves VII and VIII, respectively (Fig. 5 A, D and F). A few cells are located within this point of divergence (Fig. 5C). These latter cells were always labeled after saccular applications and, to a lesser extent, after cochlear and utricular applications. The dorsomedial cell cluster consists of very few cells (Fig. 5A and B). The axons from most of these cells first travel medially towards the floor plate, where they join the internal facial genu and turn laterally. However, a few of these cells have a similar trajectory except that they do not extend far enough medially to contribute to the internal facial genu (Fig. 5E). The ventrolateral group consists of cochlear efferent cells. This group is located ventrolaterally in the pons near the meninges (Fig. 6A and B), and forms an anterior continuation of the facial branchial motor neurons. The axons from these cells first travel dorsally, joining with the axons of the dorsolateral vestibular efferent group (Fig. 6A and B). They then turn medially and travel with the internal facial genu before turning laterally again, to exit the brain. At 14.5 dpc most ipsilateral cochlear efferent cells are located in this ventrolateral position, with the exception of a few cells that

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Fig. 4. Whole mounts of brainstems (A and C) and inner ears (B and D) demonstrating the distribution of labeled otic efferents (A-C) and afferents (D) in 12.5dpc (A) and 12 dpc (C) mice. Biotin dextran amines were applied to the right inner ear (A), the right basal plate (B and C), and to the alar plate (D). By 12,5dpc (A) labeled otic efferents are distributed bilaterally in the hindbrain and project through the VIIIth nerve roots (VIII). Note that many ipsilateral cells have already formed a laterally located longitudinal column, presumably the olivocochlear efferent group (oc). Other bilaterally labeled cells (arrowheads) remain near the floor plate (fp), or are in the process of migrating laterally (arrows). Some otic efferents have migrated into rhombomere 5. Rhombomere 5 is indicated by the level of the labeled abducens motor neurons (a), whereas contralateral otic efferent fibers cross only in rhombomere 4. Note that a half day earlier (C), applications to the right basal plate of rhombomeres 3-6 (*) label more contralateral efferent neurons than do applications to the ear and that bilaterally distributed fibers reach both eighth nerve roots (VIII). Inner ear preparations (B and D) show that in 12.0dpc mice, otic efferents(B) have traveled through the vestibular ganglion (vg), have formed the commissure of von Oort (cO), and a few fibers (arrows) extend toward the elongating epithelium of the cochlear duct (cd). Selectivelabeling of afferents (D) shows the spiral ganglion (sg) forming near the epithelium of the cochlear duct (cd) with an occasional unmigrated neuron (arrow) in the cochlear duct epithelium extending a central process that has already reached the application site in the brain. Bars = 100 pm.

are still m i g r a t i n g a n d c a n be f o u n d between the p o i n t where the facial a n d otic efferent axonal tracts diverge a n d the final p o s i t i o n of the v e n t r o l a t e r a l g r o u p (Fig. 6A, E G). T h u s by 14.5 dpc the segregation of vestibular a n d cochlear efferents is completed a n d they have nearly reached their a d u l t locations. Interestingly, the p a t t e r n of m i g r a t i o n (within secondary processes) a n d the final position (in a l o n g i t u d i n a l c o l u m n medial to the visceral m o t o r n e u r o n s ) observed in b o t h the vestibular efferent cells a n d the cochlear efferent cells are u n i q u e characteristics of m o t o r n e u r o n s derived from r h o m b o m e r e 4. As a consequence o f this m i g r a t o r y property, all m o t o r n e u r o n s derived from r h o m b o m e r e 4 either c o n t r i b u t e to the i n t e r n a l facial genu or m a k e a n a p p r o x i m a t e

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Fig. 5. Transverse 80 lam thick sections demonstrating the distribution of labeled eontralateral (A-D and F) and ipsilateral (E) vestibular efferents. DiI was applied either to the entire ear (A-C and E), or selectively to the anterior vestibular ramus of the VIIIth nerve (D and F), in mice at ages 14.5 dpc (A-D and F) and P11 (E), Midline is to the right in all sections except E. Most vestibular efferents form a dense dorsolateral cluster of cells (ve) above the otic efferent fiber tract (oe; A, D and F). Their axons join the axons of the olivocochlear efferents (oc) to form the otic efferent bundle (oe; A and C). Confluence of pathways is indicated by large arrows in A, C, D, and F. Some vestibular efferent neurons (small arrows) remain in a dorsomedial cluster near the floor plate [fp; (B)] or are located in the area where the vestibular efferent fibers separate from the olivocochlear efferent fibers (C). More laterally, some efferent branches enter the contralateral facial nerve root (f) at the point where the otic efferent fibers (oe) diverge from facial fibers (asterisk; A, D, and F). Some vestibular efferent neurons (small arrows) give offaxons that first turn medial towards the floor plate (arrowhead) and the abducens nucleus (a) and then turn lateral (open arrow) towards the facial nerve (E). Bars = 100 ~tm.

effort to d o so, w h e r e a s t h o s e f r o m r h o m b o m e r e 5 d o n o t (Fig. 2B, 3A, 4 A a n d 12). I n a d d i t i o n , all m o t o r n e u r o n s d e r i v e d f r o m r h o m b o m e r e 4 f o r m a single l o n g i t u d i n a l c o l u m n t h a t e x t e n d s f r o m r h o m b o m e r e 3 to r h o m b o m e r e 6. A l t h o u g h the s e g r e g a t i o n o f the efferent cells is c o m p l e t e at 14.5 dpc, the p a t h w a y s t a k e n b y the fibers o f m o t o r n e u r o n s d e r i v e d f r o m r h o m b o m e r e 4 are n o t fully segregated. Clearly, there is a p o i n t w i t h i n the b r a i n s t e m l a t e r a l to the facial b r a n c h i a l m o t o r / o t i c efferent cell c o l u m n w h e r e facial m o t o r a n d otic efferents diverge (Fig. 6 A a n d C). H o w e v e r , a large f r a c t i o n o f fibers, in p a r t i c u l a r after a p p l i c a t i o n s to the cochlea, e n t e r s the c o n t r a l a t e r a l facial n e r v e r o o t (Figs 5A, 6C a n d 10C).

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Fig. 6. Photomicrographs of 80 ~tm thick sections showing the distribution of contralateral (A, C, F, and G) and ipsilateral (B, D, and E) olivocochlear neurons as revealed after selective applications of DiI to the cochleas of 14.5 dpc mice. The ascending olivocochlear bundle joins (large arrow) the internal, facial genu medial to the segregation point (*) of the facial bundle (f) and the otic efferent bundle (oe) (A and C). Note the presence of numerous efferent collaterals in the contralateral facial nerve root (f) (A and C). Also note that many more cells have completed their migration and have formed the olivocochlear group (oc) on the ipsilateral side (B and D) than on the contralateral side (A and C). Many cells are in the process of migration (small arrows) on the contralateral side (A, F, and G) with many fewer migrating neurons ipsilaterally (E). Their target, the olivocochlear group, is located ventral to the migrating cells, outside the limits of the photographs in (E)-(G). Once their migration into the olivocochlear group is completed, the neurons start to develop dendritic arbors (D). Bars = 100 ~tm.

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Since no contralateral facial m o t o r neurons are labeled after these applications, this suggests that vestibular and cochlear efferents sometimes send an axon collateral into the contralateral facial nerve. We have not yet followed these fibers more peripherally to identify their termination nor do we know how long these fibers persist after birth. D I S T R I B U T I O N OF E F F E R E N T A X O N S P R O J E C T I N G TO T H E C O C H L E A We will first describe the distribution of efferents observed after dye applications to the olivocochlear bundle just lateral to the floor plate, presumably labeling all cochlear efferents. Second, this distribution will be compared to that of fibers labeled from the contralateral cochlea (bilateral cochlear efferents).

Cochlear efferent development The earliest outgrowth of cochlear efferents consists of fibers extending within the VIIIth nerve towards the spiral ganglion at 12.0 dpc. The growth cones of these first efferent fibers have almost reached the epithelium of the growing cochlear duct by this stage (Fig. 4B). By 13.5 dpc m a n y more fibers have extended toward the growing cochlea and have started to form the intraganglionic spiral bundle (Fig. 7A). At the base and the middle turn, a few collaterals branch from the intraganglionic spiral bundle and join the radial afferent fibers growing towards the developing organ of Corti. By 14.5 dpc an increasing number of efferents have joined the intraganglionic spiral bundle and even efferent fibers in the apical turn have joined the radial afferent fibers (Fig. 7B-D). By 16.5 dpc a few

Fig. 7. Whole mounts of inner ears from 13dpc (A) and 14.5dpc (B-D) mouse embryos in which the efferent fibers were selectively labeled by an application of DiI to the floor plate of rhombomere 4. At 13dpc efferents have grown through the modiolus (m) and have begun to form the intraganglionic bundle [arrowheads (A)]. The intraganglionic bundle (ig) is well formed by 14.5dpc (B-D) and efferent fibers (arrowheads) have begun to extend within radial bundles towards the organ of Corti (c) (D). Arrowheads in (C) and (D) point to the same location for orientation purposes. Bars = 100 lam.

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efferent fibers are beginning to form the outer spiral bundles in the basal (most developed), but not the apical (least developed) regions of the cochlea (Fig. 9A-C). Cross-sections through the organ of Corti reveal highly branched efferent profiles that meander extensively among the supporting cells of the greater epithelial ridge and among the inner hair cells. Interestingly, these profiles extend almost through the entire height of the sensory epithelia. A few fibers appear to extend beyond the inner hair cells, towards the outer hair cell region (Fig. 9B). By 18.5 dpc, applications of DiI to the olivocochlear bundle demonstrate that the efferents have begun to form an elaborate inner spiral bundle in the basal cochlea of some (Fig. 9D-F), but not all (Fig. 8B and D), mouse strains investigated. It appears that the cochleas of pigmented strains, such as C3H (Fig. 9A-F) and BALB/c, develop slightly faster than those of the albino strain, CFl (Fig. 8A-F), particularly in the base (Fig. SB and E). In 18.5 dpc pigmented mice, efferent branches have invaded the outer hair cell region and have arborized extensively among the outer hair cells

Fig. 8. Whole mounts of inner ears from an 185dpc mouse in which either afferents (A, C, and F) or olivocochlear efferents (B, D, and E) were selectively labeled. In the basal turn (C and D) both afferents and efferents have reached the outer hair cells. In the middle turn (A and B) afferents have reached all rows of outer hair cells and have started to form the outer spiral bundle underneath the outer hair cells in the organ of Corti (c). In contrast in the apex (E and F) afferents have started to extend to the outer hair cells while efferents are within the radial bundles (r) or are just reaching inner hair cells. Note that only spiral ganglion cells (sg) are labeled after afferent applications (A, C, and F), and only the intraganglionic fibers (ig) are labeled after selective efferent applications (B, D, and E). A&rents have formed a well-labeled inner spiral bundle (*) and inner pillar bundle (arrows) in the basal (C) and middle (D) turns before they are significantly reached by efferents (B and D). Bars = 100 urn.

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Fig. 9. Photomicrographs demonstrating the distribution of efferent fibers in the developing basal cochlea of a 16.5dpc mouse (A-C) compared to that of an 18.5dpc mouse (D-F), as revealed by photoconverted DiI applications to the olivocochlear bundle adjacent to the floorplate of the ipsilateral brainstem. Two micromiUimeter thick resin sections were stained for cellular details (A and D) or photographed with a blue filter to enhance the visibility of the DAB reaction product (B and E). Whole mounts of the basal cochlea provide an overview of efferent development at 16.5 (C) and 18.5dpc (F). Note the presence of immature inner (i) and outer hair cells (1, 2, 3). Also note that some efferents extend to the third row of outer hair cells at 16.5dpc, and form well-developedouter spiral bundles (arrowheads) paralleling the rows of outer hair cells by 18.5dpc. Efferents form a well-developed inner spiral bundle (*) and inner pillar bundle (arrows) by 18.5dpc (D and E). Their locations are indicated in the wholemount for reference purposes, although they are difficult to discern. Asterisk indicates labeled efferents located in the future inner spiral bundle. Bars = 50 lam.

(Fig. 9F), whereas in the CF1 strain efferents have very sparsely i n n e r v a t e d only the b a s a l m o s t half turn. A n extensive n e t w o r k o f efferent branches is also present a r o u n d the i n n e r hair cells, in the i n n e r spiral b u n d l e a n d i n n e r pillar b u n d l e regions (Fig. 9D a n d E). The d e v e l o p m e n t of these two b u n d l e s has previously been described by Sobkowicz a n d E m m e r l i n g . 54 Large labeled swellings below the outer hair cells indicate that efferents begin to establish contacts with outer hair cells. T h i n labeled fibers also extend t o w a r d the bases o f all three rows o f Deiter's cells.

Development of bilateral efferents Label applied to the cochlea o n one side will label efferent fibers in the c o n t r a l a t e r a l vestibulocochlear nerve, b e g i n n i n g a r o u n d 12.5 dpc. These labeled fibers are termed "bilateral efferents". Bilateral efferents first a p p e a r at almost the same time as labeled contralateral cells, a r o u n d

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12.5 d p c (Fig. 4A). These cells, like some ipsilateral cells, send a x o n s t o w a r d s the floor plate b u t they then bifurcate, with one b r a n c h p r o j e c t i n g t o w a r d each V I l l t h nerve r o o t (Fig. 4 A a n d C). T h e b r a n c h to the c o n t r a l a t e r a l V I I I t h nerve projects o n l y as far as the nerve r o o t at this time. By 14.5 dpc, the few labeled c o n t r a l a t e r a l fibers divide into two distinct b r a n c h e s u p o n e n t e r i n g the v e s t i b u l a r b r a n c h o f the V I I I t h nerve, a n a n t e r i o r a n d a p o s t e r i o r fascicle. T h e l a b e l e d fibers o f the t h i n n e r a n t e r i o r fascicle disperse as they enter the v e s t i b u l a r g a n g l i o n a n d then coalesce a g a i n distal to the g a n g l i o n to enter the v e s t i b u l a r sensory epithelia (Fig. 10A). T h e larger, p o s t e r i o r fascicle disperses a m o n g the v e s t i b u l a r g a n g l i o n cells a n d similarly coalesces a g a i n distal to the ganglion. A large f r a c t i o n o f these fibers t h e n enter the c o m m i s s u r e o f v o n O o r t to j o i n the c o c h l e a r afferent b u n d l e (Fig. 10A). A f t e r D i I a p p l i c a t i o n s restricted to the cochlea, the l a b e l e d b i l a t e r a l c o c h l e a r efferent cells projected, in m o s t cases, to the c o n t r a l a t e r a l cochlea, b u t a few b r a n c h e d into the v e s t i b u l a r g a n g l i o n a n d e n t e r e d the s a c c u l a r sensory e p i t h e l i u m (Fig. 10B). These latter b r a n c h e s could~also be labeled after D i I was selectively a p p l i e d to the saccule ( d a t a n o t shown): T h e saccular a p p l i r a t i o n s o f D i I d e m o n s t r a t e t h a t the c o c h l e a r collaterals o f these efferent fibers p r o j e c t exclus-

Fig. 10. Whole mounts of the vestibular ganglion and facial nerve (f) (A~2) and the cochlea (D), showing the distribution of contralateral efferent branches after DiI applications to the ipsilateral inner ear (A, C, and D), or the ipsilateral cochlea (B) in 14.5 dpc (A and B) and P0 mice (C and D). Arrowheads mark the edge of the vestibular ganglion. Note that efferents enter the vestibular root as two distinct fascicles that both disperse in the vestibular ganglion and then reform distally. The anterior fascicle (af) distributes efferent fibers to the utricle, saccule (s), horizontal canal, and anterior vertical canal. The posterior fascicle (pf) distributes efferents to the saccule, posterior vertical canal and the cochlea. Note that efferents to the cochlea leave the posterior fascicle proximal to the vestibular ganglion at the commissure of von Oort (cO). Selective applications to the cochlea result in the labeling of efferent branches to the contralateral saccule (s) and the contralateral facial nerve (f) at 14.5 dpc (B). Some contralateral efferent fibers extend along the intermediate nerve (in) to the geniculate ganglion on the opposite side (gg) where they join with other efferent fibers that run in the facial nerve (f) (C). The bilateral fibers that project to the contralateral ear have a similar but less extensive projection through the intraganglionic spiral bundle (ig) as compared to the projection of the ipsilateral efferent fibers (D). Bars = 100 ~tm.

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ively to the basal turn of the cochlea. 4° At birth this pattern was unchanged (Fig. 10C and D). In addition to these cochlear-saccular collaterals observed at birth, we also found a few branches of the bilateral efferents that join the intermediate nerve and extend towards the geniculate ganglion and the greater petrosal nerve (Fig. 10C). We have not yet followed the fate of these branches in neonates. As the posterior bundle of the bilateral efferents approaches the cochlea, the fibers enter the intraganglionic spiral bundle (Fig. 10D) and branch into the radial bundles. These fibers reach the inner hair cell region, but are clearly less numerous than the ipsilateral efferents (Figs 8B and 10D). It has not yet been determined if and where these fibers terminate, or even if they are maintained as the animals mature. D I S T R I B U T I O N OF A F F E R E N T AXONS P R O J E C T I N G TO T H E C O C H L E A Spiral ganglion cells emerge from the forming cochlea between 11 and 15 dpc. as Some of these cells apparently extended an axon centrally through which they could be labeled before their migration out of the cochlear anlage was complete (Fig. 4D). Thus, the spiral ganglion continues to enlarge as the cochlea grows. Afferent bundles of radial fibers form in the middle and basal turns at 14.5 dpc. At 15 dpc the last spiral ganglion cells within the apical segment become postmitotic, as and by 18.5 dpc the radial bundles have formed in the apical turn. At this age afferent fibers have extended towards the three rows of outer hair cells in the basal and the middle turns and have extended for a variable distance in the outer spiral bundles (Fig. 8A and C). This, however, is not yet the case in the apex where afferents have just started to extend beyond the inner hair cells towards the outer hair cells (Fig. 8F). In still older animals we studied the morphology of individual afferent fibers by using a small, superficial application of DiI to the cochlear nuclei. In one case we obtained 13 intensely labeled afferent fibers in a 20.5 dpc (P1) mouse embryo (Fig. 11). In the basal half of the cochlea, one fiber had a structure similar to spiral fibers (Fig. 11 3), six had structures similar to radial fibers (Fig. 1 1--2, 4, 6, 9, 10), and three had intermediate morphologies with short side branches to an inner hair cell (Fig. 11--5, 8), or branches to both inner and outer hair cells (Fig. 1 l - - l , 7). In the apical half of the cochlea, two fibers had multiple branches around one or two inner hair cells (Fig. 11 1 1, 12), and one fiber had no branches but terminated with a growth cone in the tunnel region (Fig. 11--13). The type II fiber was particularly remarkable because it could be followed from its origin in a spiral ganglion neuron to its termination as a growth cone among the outer hair cells. The cell body was basically bipolar, with asymmetrically positioned peripheral and central processes. The peripheral process gave off a single blind branch as it entered the radial fiber region. It passed through the inner hair cell region and the first two rows of outer hair cells, where it finally divided into two branches. The apical branch ended after passing five hair cells. The basal branch traveled past three hair cells and then gave off another branch which traveled apically the length of two hair cells. After passing approximately ten more hair cells, the fiber developed growth cone-like swellings and gave off numerous side branches which extended in every direction around adjacent hair cells. The side branches were typically covered with boutons. The growth cone eventually ended in two flat expansions near the base of the cochlea. DISCUSSION

Segregation of vestibulocochlear efferent neurons Our data show that rhombomere 4 gives rise to the facial-otic efferent motor complex. The neurons from this complex migrate to form a continuous column that can be segregated into three neuronal populations, the facial branchial motor neurons, the vestibular efferents, and the cochlear efferents. In contrast, rhombomere 5 gives rise to the superior salivatory nucleus (also known as facial visceral motor neurons), as well as the abducens nucleus which will not be dealt with here (Fig. 12). These neurons migrate to form a separate column lateral to the facial-otic motor complex. These data clearly argue against earlier suggestions that otic efferents are derived from visceral motor n e u r o n s . 46'47 However, the location of the otic "visceral" efferents identified by Ross in fact matches the location of the vestibular efferents identified in the present study and by others. 24'35'45

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Fig. 12. Schematic diagrams showing the distribution of facial branchial motor neurons (black), facial visceral motor neurons (gray), and otic efferents (white) and their fibers in a dorsal view of a flattened half hindbrain (11 dpc, 135 dpc) and a dorsal view of an adult brain including some peripheral nerves. At I l dpc all motor neurons of rhombomeres 4 and 5 are adjacent to the floor plate and have already extended their axons into the appropriate peripheral nerves (FB, facial branchial motor nerve; IN, intermediate nerve; VIII, eighth nerve). At 13.5dpc facial branchial motor neurons (black) have migrated along the floor plate, through rhombomere 5 and into rhombomere 6. Their trailing axons form part of the internal facial genu of rhombomeres 4 and 5. Visceral motor neurons have migrated laterally and do not contribute to the internal facial geuu. In contrast, otic efferents, which have also migrated laterally, send their axons into the internal facial genu and develop axonal collaterals that cross the floor plate in rhombomere 4 (small white arrow). As a consequence of their migration patterns, facial branchial motor neurons and otic efferent neurons form an almost continuous column of cells medial to the visceral efferents of the superior salivatory nucleus. The facial branchial motor fibers extend only through the facial nerve to the periphery. In contrast visceral motor neurons, which join facial branchial motor axons via the intermediate nerve diverge to different targets through the greater petrosal (GP), and chorda tympani nerves (CT). Otic efferent neurons are the only population that develop contralaterally projecting fibers, all of which cross in rhombomere 4. Peripherally, otic efferents contribute to the anterior (VIIIa) and posterior (VIIIp) rami of the eighth nerve.

The location of the facial visceral (superior salivatory) m o t o r n e u r o n s in adults j° is c o m p a r a b l e to the location we have described for embryos, a n d is clearly lateral to the visceral m o t o r n e u r o n area identified by Ross. 47 However, our data showing that some a b e r r a n t otic efferent fibers travel in the i n t e r m e d i a t e nerve suggests that otic efferents m a y rarely follow the p a t h w a y of visceral m o t o r n e u r o n s t h o u g h derived from a different r h o m b o m e r e . In a d d i t i o n to their c o m m o n origin, the facial b r a n c h i a l a n d otic efferent m o t o r n e u r o n s have similar a x o n a l trajectories, whereas the facial visceral m o t o r n e u r o n s have both a separate a x o n a l trajectory to exit the brain, a n d a separate r h o m b o m e r e of origin (Fig. 12). G u i n a n e t al., 25 showed that different facial b r a n c h i a l m o t o r s u b p o p u l a t i o n s have c o m m o n a x o n a l trajectories t h r o u g h the internal facial genu, a n d suggested that these p o p u l a t i o n s of cells have similar embryologic origins near the i n t e r n a l facial genu. O u r results are consistent with this hypothesis. Likewise, B r o w n 3 showed that the axons of b o t h medial a n d lateral olivocochlear efferents project t h r o u g h the internal facial genu, t h o u g h only the medial olivocochlear efferents were previously t h o u g h t to do so. 42 44.61 The similar a x o n a l p a t h w a y t h r o u g h the i n t e r n a l facial genu for b o t h the facial b r a n c h i a l m o t o r n e u r o n s a n d otic efferents, b u t n o t for the visceral m o t o r n e u r o n s can n o w be explained based o n the origin a n d s u b s e q u e n t m i g r a t i o n of these cells: the course of their axons t h r o u g h the genu reflects the m i g r a t i o n route of all m o t o r n e u r o n s derived from r h o m b o m e r e 4 (Fig. 12). In contrast, the m o t o r n e u r o n s of r h o m b o m e r e 5 translocate within their axons until they reach a p o i n t lateral to the future branchial/efferent c o l u m n , from which they translocate in a secondary process to their final position. Thus, o u r d e v e l o p m e n t a l analysis has clarified several issues that r e m a i n e d unresolved based on a d u l t descriptions. Moreover, our d a t a fully agree with recent studies using superior m o d e r n tracing techniques to establish that the p a t h w a y s taken by the axons o f m o t o r n e u r o n s within the h i n d b r a i n are indicative of their origins a n d m i g r a t i o n patterns. 3"25

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Although others have reported the distribution of facial motor neurons, otic efferents and visceral motor neurons in young embryos, 8A6'17'36 to our knowledge a complete account illustrating their segregation into four discrete clusters (facial branchial motor neurons, facial visceral motor neurons, olivocochlear efferents and vestibular efferents) and their projections to discrete nerves and targets through prenatal and early postnatal development have not been previously published. Several other accounts of the early distribution of motor neurons in rhombomeres 4 and 5 agree with our data. 22'23'3° However, their interpretations differ from ours with respect to the origins of the otic efferents and visceral motor neurons, largely because they ignore visceral motor neurons and assign various labels to the motor neurons of rhombomere 5. For example, two recent papers 22'3° have suggested that otic efferents are derived from rhombomeres 4 and 5 in mice. This interpretation is in contrast to other, earlier interpretations from the same laboratory suggesting they are derived exclusively from rhombomere 4. 36 Furthermore, our interpretation that the facial visceral motor neurons are derived from rhombomere 5 is supported by data gathered in Kreisler mutants. 19 Rhombomere 5 does not form in these mutants, 11 and both the abducens nucleus and the facial visceral motor (superior salivatory) nucleus are completely absent. ~9 In summary, several long-standing issues about the relationships of vestibular, cochlear, facial branchial, and visceral motor neurons have been resolved. However, the origins of the facial branchial motor neurons and otic efferents from a different rhombomere (rhombomere 4) than the visceral motor neurons (rhombomere 5) in mice contrasts with the origins of these groups in other vertebrates. For example, in chickens the facial branchial and otic motor neurons originate from rhombomeres 4 and the rostral half of rhombomere 5, and visceral motor neurons from the caudal half of rhombomere 5,16 and in lampreys it is unclear whether visceral motor neurons exist at all. 18 Thus the segregated origins of morphologically distinct facial-otic motor neurons and visceral motor neurons from rhombomeres 4 and 5, respectively, represent an evolutionary novelty unique to mammals. Development of efferent innervation to the cochlea Our data clearly demonstrate that the outgrowth of efferents towards the hair cells is essentially completed in mice before birth and before the onset of function. 32'6° Thus, the mechanisms that govern this fiber outgrowth and produce an initial crude tonotopic map of efferents in the cochlea must use cues other than functional activity to direct the growth of efferent (and afferent) fibers. It appears that specific activity will, at best, help to focus the generalized topological pattern that was laid down in the embryo and transform it into the refined pattern observed in adult mice. TM Two major classes of efferents have been observed in adult mammals: small unmyelinated axons that terminate as fine spiral fibers beneath inner hair cells, and large axons that synapse on outer hair cells. 2'3'6z An additional class of efferents with synapse-like contacts on Deiter's cells has been observed in neonatal humans and r a t s Y 9 The development of the efferents has been studied with labeling techniques in hamsters, mice, rats, and c a t s 5'9"41'51'63 revealing a fairly similar sequence of events, although their precise timing varies considerably among species. In mice, efferents have reached the vicinity of inner hair cells and some outer hair cells by 16 dpc, and have formed inner and outer spiral bundles by 18 dpc. In most rodents these events happen shortly before or after the time of b i r t h , 5'26'28'29'31'37'38'5~52"63although in animals with longer gestation periods such as guinea pigs, efferent and afferent synaptogenesis is in progress well before birthY The developing efferents first give rise to branches that terminate on or near the inner hair cells. After this initial phase, many efferents terminate on both inner hair cells and outer hair cells, thus forming an intermediate class between the two adult classes, 4~'51 and other efferents terminate only on inner hair cells or only on outer hair cells, similar to the efferents observed in a d u l t s . 2'3'62 Efferent projections to outer hair cells tend to arborize extensively among more than one row initially, and then to restrict their arbor to a single row of cells. 63 Efferent axons cross to the outer hair cell region before the tunnel of Corti opens (Fig. 9A and B). 5 Electron microscopy of developing efferents demonstrates that efferents are virtually indistinguishable from afferents without selective labeling. 5'14'52'54'56'5vImmature efferents contacting both inner and outer hair cells contain few synaptic vesicles, and may be apposed by a variety of postsynaptic specializations, including postsynaptic cisterns, one or more vesicles of various sizes, and occasional synaptic ribbons without clear-cut presynaptic thickeningsY T M As efferent synapses

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mature, they acquire specializations that clearly distinguish them from afferents, c.~.57 We now need to elucidate the interactions that occur between the efferent growth cone and the hair cell and how closely they mimic the temporal and molecular development of the neuromuscular junctions formed by facial branchial motor neurons. ~2

Development of afferent innervation to the cochlea Our data on the afferents confirm earlier descriptions based on silver stains, 6'33'53'55'58and extend them with a more detailed analysis of the growth of selectively labeled afferents. Cajal, 6 Lorente de N633 and Tello 5s labeled both afferent and efferent fibers with the silver stain. Afferent cochlear innervation was finally distinguished from efferent innervation when the silver stain was applied to tissue cultures that contained exclusively afferent fibers, s3's5 Figures 21, 22 and 27 of Sobkowicz and Rose 55are particularly noteworthy, because they show that afferent fibers between the spiral ganglion and the organ of Corti are all distributed radially, with no intraganglionic fibers. The major findings of these earlier studies were remarkably accurate given the techniques available. The selective DiI labeling in the present study does, however, demonstrate a previously unrealized degree of early differentiation of type II neurons projecting to the outer hair cells. Previously it was shown that type I I afferents (the deep radial fibers of Tello) 58,just begin to extend to outer hair cells by 16.5 dpc. In contrast, our data show that at this age the growing type II neurites have already reached the outer hair cells of the basal and middle turns and have started to form outer spiral bundles. Although silver stains selectively stain neurofilaments, growth cones have relatively few neurofilaments and so would be poorly impregnated. Therefore, the extent of afferent growth was probably underestimated in studies using silver stains. It is apparent in our preparations that by 18.5 dpc some afferents have already extended into the third row of outer hair cells, whereas earlier descriptions, probably due to an incomplete impregnation of the growing fiber tips, suggest that this occurs near the time of birth. 5558 Clearly, more data with restricted applications of DiI, followed by photoconversion are needed to clarify these issues further. The development of labeled afferent projections to hair cells has been studied in a variety of species. 5'~3'4~52 Studies in neonatal kittens, gerbils, and mice demonstrate that early in development many afferents branch to both inner and outer hair cells. 5~3'4~'52 Within a few days these arbors are reduced until they innervate either the inner or outer hair cells, but not both. t3 However, some investigators have identified a third, possibly transient, type of spiral ganglion cell that contributes fibers to the inner spiral bundle for a short distance. 52 The afferents projecting to inner hair cells first arborize among several adjacent inner hair cells, and later become restricted to a single inner hair cell. The afferents projecting to outer hair cells spiral in the outer spiral bundles towards the cochlear base, and form increasing numbers of branches towards the termination of their spiral. Our results in mice demonstrate that these various states of afferent development are present at 20 dpc in a loose basal to apical gradient in which afferent types restricted to inner or outer hair cells are more frequent basally, and those with dual innervation or a single growth cone, are more frequent apically. Similarly, cultures of newborn mouse cochlea develop comparable afferent arbors, and also suggest that afferent fibers may be capable of bifurcating and innervating inner hair cells located as far as 600 gm apart. 52 Likewise, ganglion cells near the apex may send collaterals through more than one radial bundle. However, the lack of aberrant branching by developing afferents observed in neonates by Echteler ~3and us, suggests that growing afferents do not use massive arborization to find their appropriate target, but are directed to the appropriate hair cell region with relatively little error once they have entered a given radial bundle. Together, these results suggest that more-or-less specific pathfinding mechanisms operate within the developing limbus rather than a simple attraction towards hair cells. Finally, in spite of the work done in this area, little is known about the growth and development of afferents before they reach the inner hair cell region, such as the choice of which radial bundle to enter. Afjerent and efJerent interactions in the developing cochlea A widely accepted view of the development of cochlear innervation is that afferent fibers innervate hair cells before efferents, and that the efferents subsequently compete with the afferents for available

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contact sites at inner and outer hair cells. 31'42 This view was based on material in which afferents and efferents were not selectively labeled. However, the results of more recent investigations using experimental labeling such as immunocytochemistry and histochemical reactions has cast some doubt on this scheme because they indicate that in mice the efferents arrive prior to birth. 52'6°Thus several modifications to the current view are warranted. First, both afferents and efferents have contacted inner and outer hair cells before birth, and synaptogenesis is well underway before the onset of hearing. Second, the general topological pattern of efferent and afferent innervation is also established before the onset of hearing. In the mouse, the organ of Corti is innervated prenatally and neurites can be seen in the hair cell region as early as 12 dpc (Fig. 4D). 21'34'58 Specifically, the afferents reach the hair cell region as early as 12 dpc, although there is some variation among mouse strains. 2°'58 Efferents reach the hair cell region as early as 16.5 dpc based on histochemical detection of acetycholine esterase. 52'54The results of the present study demonstrate that efferents reach the hair cell region by 12 dpc (Figs 4 and 7A) and may come in contact with hair cells as early as 14.5 dpc (Fig. 7C and D). In direct comparisons of efferent and afferent progression (Fig. 4B and D, and 8A-F), it is obvious that efferents are lagging only slightly behind afferents at any given age. For example, in the CF1 mouse strain, afferents in the middle cochlear turn have already formed the outer spiral bundles by 18.5 dpc, but efferents do not contribute to the outer spiral bundle although they have reached all rows of hair cells (Fig. 8A and B). Thus, growing efferents appear to follow along afferents in proximal and distal spiral ganglion processes, suggesting that afferents may function as a substrate that efferents grow along. However, efferents disperse once they reach both the vestibular and the cochlear ganglia (Fig. 10A and C), only to coalesce again once they pass the ganglia, and then form the intraganglionic spiral bundle. The substrates along which efferents extend in these regions need to be investigated. One possibility is that efferent fibers use the afferent subepithelial plexus at the edge of the spiral ganglion,6'55as a substrate for growth (Sobkowicz, personal communication), yet other factors must also be involved to direct the efferent growth into a basal to apical spiral and to enter particular radial bundles. It was recently shown in mice with neurotrophin receptor knockouts that efferents do not project to sensory epithelia that lack afferent innervation, suggesting again that efferents may need afferents to grow along. 2° Our data conflict with this suggestion only with respect to the efferent dispersal within the ganglia and intraganglionic spiral bundle. Apparently the ganglia have different effects on efferent growth than on either proximal or distal afferent ganglionic processes. Precise comparisons of developmental stages observed in various studies are difficult because the gestation will vary from 18 to 21 days among different strains. 49 Similarly, the rate of development will vary among fetuses of the same litter depending on their positions in the uterine horns. Finally, developmental stage will depend on which turn of the cochlea is selected for study, since there is a base to apex gradient of as much as 4 days. 58 Although our data suggest a much earlier arrival of afferents and efferents at the developing hair cells than previously suggested, they do not necessarily contradict the numerous papers dealing with the reorganization of both afferent and efferent fibers at cochlear hair cells. In fact, the limited number of afferents investigated show that they develop much larger terminal arbors in neonates than are present in older animals. Thus pruning of excessive collaterals of afferents~3"52may in fact occur in neonates, and may in part be governed by a number of molecules recently characterized in the developing cochlea) 8'29'6° Because our data strongly suggest that this reorganization starts prenatally, investigations of topographic development and the expression of these molecules need to be extended to these earlier stages. Our findings on prenatal arrival of fibers as well as onset of synaptogenesis in mice are consistent with the data from other species. In rats, for example, spectrin immunolabeling in the basal turn of the cochlea, which selectively stains efferents, labels fibers outside the greater epithelial ridge at 18 dpc and below the inner hair cell region at 20 dpc. 26'6° Efferents have clearly reached the inner and outer hair cell regions by the day of birth in rats? '26~6°Thus in rats the efferents reach the hair cell regions at a slightly older age than in mice, but this nonetheless occurs prenatally in both species. Likewise, guinea pig efferents form mature synapses on hair cells several days before birth and before the onset of sound perception through the middle ear mechanism.27 Thus, in these animals most of the topologically restricted representation of both afferents and efferents will be developed before any adult-like tonotopic representation of frequencies occurs in the cochlea. Together these data stress that most of the guidance cues that establish the topological projection patterns of

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afferents and efferents during development must rely on principles other than patterned activity, although patterned activity may be necessary to fine-tune connections. Acknowledgements--We thank Maria Christensen and Carole Miller for their excellent histological and photographic

expertise. We are grateful for the insightful comments provided by two anonymous referees. Supported by NIH Program Project Grant P50-DC00215 to LLB, DHN, and BF.

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